Manipulation of genes tagged with recombinant transposons with transposase and site-specific DNA recombinases permits mutagenesis and gene replacement of genomic loci (Rubin & Spradling 1982; O'Kane & Gehring 1987; Kawakami et al. 2000; Bischof et al. 2007; Venken et al. 2011). Although these technologies have considerably advanced molecular genetic studies in Drosophila, their application is still limited by incomplete coverage of transposons insertions. Designable site-specific DNA endonucleases have greatly expanded the opportunity of gene manipulation to untagged loci. The modular nature of nucleotide recognizing domains of zinc-finger type (Carroll 2011) or TAL effector type (Joung & Sander 2012) permits systematic design of specific DNA binding proteins. Pairs of such proteins recognizing nearby sequences of loci of interest are fused to Fok I DNA endonuclease. Properly designed pairs of such fusion proteins dimerize to form active nuclease and introduce double strand DNA breaks at specific genomic loci, and induce DNA recombination and deletion through non-homologous end joining (NHEJ). Although these strategies were well received, relatively few cases of actual use on Drosophila have been reported (Beumer et al. 2008; Katsuyama et al. 2013; Sakuma et al. 2013).
Here we applied the technique of TALEN on mutagenesis of the trachealess (trh) gene in Drosophila, which is located at the tip of the left arm of chromosome 3 (61C1). Although mutant alleles have been available, they are either missense mutation (Jürgens et al. 1984; Chung et al. 2011) or insertion/deletion at 5′ non-coding region (Isaac & Andrew 1996; Wilk et al. 1996), and no molecularly defined protein null mutation has been available. We report design and procedures of TALEN based gene knockout of trh and discuss the potential and the advantage of this method compared to other methods of gene mutagenesis in Drosophila.